Method for the production of biomass from plant differentiated tissue

ABSTRACT

The present invention provides a method and a culture medium for the production of food biomass by directly culturing seed kernel tissue, or seed cotyledonary differentiated tissue. The culture medium of the present invention contains at least DKW culture medium, Vitamin MS culture medium with an enriched concentration of thiamine, sacarose, kinetine, adenine, 2,4 d iclorofenoxiacético (2,4D), L-Glutamine, cysteine, ascorbic acid, and Gelrite®.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/613,865, filed Dec. 20, 2006, pending, of which the entire contentsare herein incorporated by reference.

FIELD OF THE INVENTION

The invention is related to a method and a culture medium for theproduction of food biomass from plant seed differentiated tissue.

BACKGROUND OF THE INVENTION

The U.S. Pat. No. 4,204,366 by Janick, Jules et al. describes a methodfor the production of cotyledons by non-agricultural means. However, themethod described by Janick requires propagating or culturing asexualembryos. Regeneration of plants by somatic embryogenesis has also beendescribed by Sondahl, M. R. et al (U.S. Pat. No. 5,312,801)

The present invention provides a culture medium and a method to producefood biomass by directly culturing seed differentiated and mature tissuewithout propagating or culturing embryos.

SUMMARY OF THE INVENTION

The invention of the present application provides a non-agriculturalmethod good for vegetal food production, wherein the method does notrequire growing cacao plants, clonal propagation of plants byembryogenesis, or production of cotyledons by embryogenesis. Inaddition, the method of the present invention can be scaled up in alaboratory or bioreactor without worrying about weather conditions orcontamination by virus or plagues. This new non-agricultural method forvegetal food biomass production is a new answer to the increasing needof food for the world population

The object of the present invention is to provide a method and a culturemedium for the production of biomass by directly culturing seed kernel,or seed cotyledonary differentiated tissue.

The culture medium of the present invention contains at least DKWculture medium, Vitamin MS culture medium with an enriched concentrationof thiamine, sacarose, kinetine, adenine, 2,4diclorofenoxiacético(2,4D), L-Glutamine, cysteine, ascorbic acid, and Gelrite®.

Specifically, the present invention provides a method and culture mediafor the production of biomass from seed cotyledonary differentiatedtissue or seed kernel from plants from the Sterculiacea and Proteaceaefamilies respectively.

Objectives and additional advantages of the present invention willbecome more evident in the description of the figures, the detaileddescription of the invention and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a Theobroma cacao (Sterculiacea family)plant seed and a representation of the portion where the sample ofcotyledonary differentiated tissue is taken from, to culture for theproduction of cacao biomass.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to provide a culture medium forthe production of biomass from plant seed differentiated and maturetissue comprising at least DKW culture medium, Vitamin MS culture mediumwith an enriched concentration of thiamine, sacarose in a concentrationbetween 15 and 40 g/L, kinetine in a concentration between 0.5 and 4mg/L, adenine in a concentration between 0.2 and 5 mg/L,2,4diclorofenoxiacético (2,4D) in a concentration between 1 and 5 mg/L,L-Glutamine in a concentration between 20 and 100 mg/L, cysteine in aconcentration between 10 and 100 mg/L, ascorbic acid in a concentrationbetween 20 and 110 mg/L, and Gelrite® in a concentration between 0.7 and2.0 g/L, wherein the culture medium has the pH adjusted between 5.5 and6.0.

In a prefer embodiment, the culture medium of the present inventioncomprises at least agar, DKW culture medium, Vitamin MS (Murashige andSkoog) culture medium with an enriched concentration of thiamine,sacarose in a concentration of 20 g/L, kinetine in a concentration of 2mg/L, adenine in a concentration of 1 mg/L, 2,4diclorofenoxiacético(2,4D) in a concentration of 4 mg/L, L-Glutamine in a concentration of50 mg/L, cysteine in a concentration of 20 mg/L, ascorbic acid in aconcentration of 20 mg/L, and Gelrite® in a concentration of 1.6 g/L,wherein the culture medium has the pH adjusted to 5.8, and wherein theDKW culture medium and the Vitamin MS culture medium concentrations perliter are prepared according to the suggested standard commercialguidelines.

FIG. 1 is an illustration of the parts of a Theobroma cacao seed: seedcoat (1); cotyledonary differentiated tissue (2); and embryo (3).

FIG. 1 also shows a representation of one aspect of the presentinvention where a portion sample of seed differentiated and maturetissue (4)(shaded area) is cotyledonary differentiated tissue (3). Inthis aspect of the invention the seed differentiated and mature tissueis from a plant from the Sterculiacea family. In a preferred embodimentof this aspect of the present invention, the plant from the Sterculiaceafamily is from the Theobroma genus. Yet, in a more preferred embodimentof this aspect of the present invention the plant from the Sterculiaceaplant is from the T. cacao species.

In another aspect of the present invention, the plant seeddifferentiated and mature tissue is seed kernel, wherein the plant seeddifferentiated tissue is from a plant from the Proteaceae family. In apreferred embodiment of this another aspect of the present invention,the plant from the Proteaceae family is from the Macadamia genus. Yet,in a more preferred embodiment of this another aspect of the presentinvention, the plant from the Proteaceae family is from the Macadamiaspecies.

In addition, the second object of the present invention is to provide amethod for the production of biomass from Sterculiacea plantdifferentiated and mature tissue comprising:

-   -   A. Obtaining a fruit that is between three and five months old;    -   B. Cleaning and sterilizing said fruit;    -   C. Extracting seeds from said fruit;    -   D. Discarding the embryo and obtaining a sample of cotyledonary        differentiated tissue;    -   E. Putting the sample of cotyledonary differentiated tissue in        contact with culture medium;    -   F. Incubating under light with a determined wave length, and at        a temperature between 15 and 35° C., until biomass is produced;        and,    -   G. Collecting grown biomass tissue by retiring said grown        biomass tissue from the culture medium.

Wherein in one aspect of the method of the present invention the culturemedium contains at least DKW culture medium, Vitamin MS culture mediumwith an enriched concentration of thiamine, sacarose in a concentrationbetween 15 and 40 g/L, kinetine in a concentration between 0.5 and 4mg/L, adenine in a concentration between 0.2 and 5 mg/L,2,4diclorofenoxiacético (2,4D) in a concentration between 1 and 5 mg/L,L-Glutamine in a concentration between 20 and 100 mg/L, cysteine in aconcentration between 10 and 100 mg/L, ascorbic acid in a concentrationbetween 20 and 110 mg/L, and Gelrite® in a concentration between 0.7 and2.0 g/L; and wherein the pH of said culture medium is adjusted to arange between 5.5 and 6.0. However, in a preferred embodiment of thisaspect of the method of the present invention, the culture mediumcontains at least agar, DKW culture medium, Vitamin MS (Murashige andSkoog) culture medium with an enriched concentration of thiamine,sacarose in a concentration of 20 g/L, kinetine in a concentration of 2mg/L, adenine in a concentration of 1 mg/L, 2,4diclorofenoxiacético(2,4D) in a concentration of 4 mg/L, L-Glutamine in a concentration of50 mg/L, cysteine in a concentration of 20 mg/L, ascorbic acid in aconcentration of 20 mg/L, and Gelrite® in a concentration of 1.6 g/L;and the pH of the culture medium is adjusted to 5.8.

In one more aspect of the method of the present invention, thecotyledonary differentiated and mature tissue is from a Sterculiaceaplant from the Theobroma genus. Still, in a preferred embodiment of thisaspect of the present invention the Sterculiacea plant is from the T.cacao species.

In a preferred embodiment of another additional aspect of the method ofthe present invention the sample of cotyledonary differentiated andmature tissue in contact with the culture medium is incubated undertotal darkness.

Moreover, the third object of the present invention is to provide amethod for the production of biomass from Proteaceae plantdifferentiated and mature tissue comprising:

-   -   a. Obtaining a sample of kernel differentiated tissue;    -   b. Putting the sample of kernel differentiated tissue in contact        with culture medium;    -   c. Incubating under light with a determined wave length, and at        a temperature between 15 and 35° C., until biomass is produced;        and,    -   d. Collecting grown biomass by retiring said grown biomass from        the culture medium.

Wherein in one aspect of the method for the production of biomass fromProteaceae plant differentiated and mature tissue, the culture mediumcontains at least DKW culture medium, Vitamin MS culture medium with anenriched concentration of thiamine, sacarose in a concentration between15 and 40 g/L, kinetine in a concentration between 0.5 and 4 mg/L,adenine in a concentration between 0.2 and 5 mg/L,2,4diclorofenoxiacético (2,4D) in a concentration between 1 and 5 mg/L,L-Glutamine in a concentration between 20 and 100 mg/L, cysteine in aconcentration between 10 and 100 mg/L, ascorbic acid in a concentrationbetween 20 and 110 mg/L, and Gelrite® in a concentration between 0.7 and2.0 g/L; and wherein the pH of said culture medium is adjusted to arange between 5.5 and 6.0. However, in a preferred embodiment of thisaspect of the method of the present invention, the culture mediumcontains at least agar, DKW culture medium, Vitamin MS (Murashige andSkoog) culture medium with an enriched concentration of thiamine,sacarose in a concentration of 20 g/L, kinetine in a concentration of 2mg/L, adenine in a concentration of 1 mg/L, 2,4diclorofenoxiacético(2,4D) in a concentration of 4 mg/L, L-Glutamine in a concentration of50 mg/L, cysteine in a concentration of 20 mg/L, ascorbic acid in aconcentration of 20 mg/L, and Gelrite® in a concentration of 1.6 g/L;and the pH of the culture medium is adjusted to 5.8.

In one more aspect of the method for the production of biomass fromProteaceae plant differentiated and mature tissue, the seed kerneldifferentiated tissue is from a Proteaceae plant from the Macadamiagenus. Still in a preferred embodiment of this aspect of this method,the Proteaceae plant is from the Macadamia species.

In a preferred embodiment of another additional aspect of the method forthe production of biomass from Proteaceae plant differentiated andmature tissue, the sample of kernel differentiated tissue in contactwith the culture medium is incubated under total darkness.

The method and culture of the present invention could also be appliedfor the production of biomass from seed differentiated tissue from othernut plants, as for example; almonds, walnuts, cashew nuts, Brazil nuts,hazelnuts, chestnuts, chickpeas, pistachios, pecan nuts, pine nuts,peanuts, heart nuts, persina walnuts, etc.

While the description presents the preferred embodiments of the presentinvention, additional changes can be made in the form and disposition ofthe parts without distancing from the basic ideas and principlescomprised in the claims.

EXAMPLE Materials and Methods

The culture medium for production of biomass was prepared as follows:

Agar, DKW culture medium, Vitamin MS (Murashige and Skoog) culturemedium with an enriched concentration of thiamine, sacarose 20 g/L,kinetine 2 mg/L, adenine 1 mg/L, 2,4diclorofenoxiacético (2,4D) 4 mg/L,L-Glutamine 50 mg/L, cysteine 20 mg/L, ascorbic acid 20 mg/L, andGelrite® 1.6 g/L. The culture medium has the pH adjusted to 5.8, and theDKW culture medium and the Vitamin MS culture medium concentrations wereprepared according to the suggested standard commercial guidelines.

Three to four month old cacao fruits were obtained from a Colombianfarm. The cacao fruits were cleaned and sterilized. The seed from saidfruits were extracted. The embryos from the cacao seed were discarded.Samples of approximately 5 mm from the cotyledonary differentiatedtissue were cut from the seed. The cotyledonary differentiated tissuesamples were put in Petri plates with the culture medium. The cultureplates with the cotyledonary differentiated tissue samples wereincubated under darkness at room temperature for 1-3 months. The newgrown dark brown biomass tissue buds were collected by separating itfrom the solid culture medium. The dark brown tissue buds were splitinto several pieces and each was re-inoculated into a new Petri platewith the culture medium. After approximately one month, the new darkbrown tissue buds were collected again.

The collected dark brown tissue buds have a cacao flavor when orallytasted. In addition, preliminary analysis of the dark brown tissue budsshows that they contain at least cacao butter and cacao carbohydrates.

Fresh mature macadamia fruits were obtained in a local market. Themacadamia fruits were cleaned and sterilized. Inside a flow chamber,samples of approximately 5 mm from the kernel differentiated and maturetissue of the fruit were cut. The kernel differentiated and maturetissue samples were put in Petri plates with the culture medium. Theculture plates with the kernel differentiated and mature tissue sampleswere incubated under darkness at room temperature for 1-3 months. Thenew grown whitish creamy biomass tissue buds were collected byseparating it from the solid culture medium. The whitish creamy biomasstissue buds were split into several pieces and each piece wasre-inoculated into a new Petri plate with culture medium. Afterapproximately one month, the new whitish creamy biomass tissue buds werecollected again.

The collected whitish creamy biomass tissue buds from macadamia beanswas macroscopically analyzed with a light microscope, in which, fattytissue was observed. This initial observation suggests that said biomasscontains at least macadamia fat components. In addition, the whitishcreamy biomass tissue buds have macadamia flavor when orally tested.

1. A method for the production of biomass from Sterculiacea plantdifferentiated tissue comprising: A. Obtaining a fruit that is betweenthree and five months old; B. Cleaning and sterilizing said fruit; C.Extracting seeds from said fruit; D. Discarding the embryo and obtaininga sample of cotyledonary differentiated tissue; E. Putting the sample ofcotyledonary differentiated tissue in contact with culture medium; F.Incubating under light with a determined wave length, and at atemperature between 15 and 35° C., until biomass is produced; and, G.Collecting grown biomass by retiring said grown biomass from the culturemedium.
 2. The method of claim 1, wherein the culture medium contains atleast DKW culture medium, Vitamin MS culture medium with an enrichedconcentration of thiamine, sacarose in a concentration between 15 and 40g/L, kinetine in a concentration between 0.5 and 4 mg/L, adenine in aconcentration between 0.2 and 5 mg/L, 2,4diclorofenoxiacético (2,4D) ina concentration between 1 and 5 mg/L, L-Glutamine in a concentrationbetween 20 and 100 mg/L, cysteine in a concentration between 10 and 100mg/L, ascorbic acid in a concentration between 20 and 110 mg/L, andGelrite® in a concentration between 0.7 and 2.0 g/L; and wherein the pHof the culture medium is adjusted to a range between 5.5 and 6.0.
 3. Themethod of claim 1, wherein the Sterculiacea plant is from the Theobromagenus.
 4. The method of claim 1, wherein the Sterculiacea plant is fromthe T. cacao species.
 5. The method of claim 1, wherein the sample ofcotyledonary differentiated tissue in contact with the culture medium isincubated under total darkness.
 6. A method for the production ofbiomass from Proteaceae plant differentiated tissue comprising: a.Obtaining a sample of kernel differentiated tissue; b. Putting thesample of kernel differentiated tissue in contact with culture medium;c. Incubating under light with a determined wave length, and at atemperature between 15 and 35° C., until biomass is produced; and, d.Collecting grown biomass by retiring said grown biomass from the culturemedium.
 7. The method of claim 6, wherein the culture medium contains atleast DKW culture medium, Vitamin MS culture medium with an enrichedconcentration of thiamine, sacarose in a concentration between 15 and 40g/L, kinetine in a concentration between 0.5 and 4 mg/L, adenine in aconcentration between 0.2 and 5 mg/L, 2,4diclorofenoxiacético (2,4D) ina concentration between 1 and 5 mg/L, L-Glutamine in a concentrationbetween 20 and 100 mg/L, cysteine in a concentration between 10 and 100mg/L, ascorbic acid in a concentration between 20 and 110 mg/L, andGelrite® in a concentration between 0.7 and 2.0 g/L; and wherein the pHof the culture medium is adjusted to a range between 5.5 and 6.0.
 8. Themethod of claim 6, wherein the Proteaceae plant is from the Macadamiagenus.
 9. The method of claim 6, wherein the Proteaceae plant is fromthe Macadamia species.
 10. The method of claim 6, wherein the sample ofkernel differentiated tissue in contact with the culture medium isincubated under total darkness.